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1.
Cancer Cell ; 35(5): 738-751.e9, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31085175

RESUMO

Ripretinib (DCC-2618) was designed to inhibit the full spectrum of mutant KIT and PDGFRA kinases found in cancers and myeloproliferative neoplasms, particularly in gastrointestinal stromal tumors (GISTs), in which the heterogeneity of drug-resistant KIT mutations is a major challenge. Ripretinib is a "switch-control" kinase inhibitor that forces the activation loop (or activation "switch") into an inactive conformation. Ripretinib inhibits all tested KIT and PDGFRA mutants, and notably is a type II kinase inhibitor demonstrated to broadly inhibit activation loop mutations in KIT and PDGFRA, previously thought only achievable with type I inhibitors. Ripretinib shows efficacy in preclinical cancer models, and preliminary clinical data provide proof-of-concept that ripretinib inhibits a wide range of KIT mutants in patients with drug-resistant GISTs.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Animais , Células CHO , Linhagem Celular , Linhagem Celular Tumoral , Cricetulus , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/genética , Células HCT116 , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Mutação/efeitos dos fármacos , Mutação/genética
2.
Mol Cancer Ther ; 14(9): 2023-34, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26285778

RESUMO

Altiratinib (DCC-2701) was designed based on the rationale of engineering a single therapeutic agent able to address multiple hallmarks of cancer (1). Specifically, altiratinib inhibits not only mechanisms of tumor initiation and progression, but also drug resistance mechanisms in the tumor and microenvironment through balanced inhibition of MET, TIE2 (TEK), and VEGFR2 (KDR) kinases. This profile was achieved by optimizing binding into the switch control pocket of all three kinases, inducing type II inactive conformations. Altiratinib durably inhibits MET, both wild-type and mutated forms, in vitro and in vivo. Through its balanced inhibitory potency versus MET, TIE2, and VEGFR2, altiratinib provides an agent that inhibits three major evasive (re)vascularization and resistance pathways (HGF, ANG, and VEGF) and blocks tumor invasion and metastasis. Altiratinib exhibits properties amenable to oral administration and exhibits substantial blood-brain barrier penetration, an attribute of significance for eventual treatment of brain cancers and brain metastases.


Assuntos
Aminopiridinas/farmacologia , Anilidas/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neovascularização Patológica , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Receptor TIE-2/antagonistas & inibidores , Microambiente Tumoral , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Aminopiridinas/química , Anilidas/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Bevacizumab/química , Bevacizumab/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Desenho de Fármacos , Quimioterapia Combinada , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Concentração Inibidora 50 , Melanoma Experimental , Camundongos , Modelos Moleculares , Conformação Molecular , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptor TIE-2/metabolismo , Proteínas Recombinantes , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
3.
J Biomol Screen ; 16(7): 724-33, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21521800

RESUMO

Transforming growth factor ß (TGF-ß) type I receptor (activin receptor-like kinase 5, ALK5) has been identified as a promising target for fibrotic diseases. To find a novel inhibitor of ALK5, the authors performed a high-throughput screen of a library of 420,000 compounds using dephosphorylated ALK5. From primary hits of 1521 compounds, 555 compounds were confirmed. In total, 124 compounds were then selected for follow-up based on their unique structures and other properties. Repeated concentration-response testing and final interference assays of the above compounds resulted in the discovery of a structurally novel ALK5 inhibitor (compound 8) (N-(thiophen 2-ylmethyl)-3-(3,4,5 trimethoxyphenyl)imidazo[1,2ß]pyridazin 6-amine) with a low IC(50) value of 0.7 µM. Compound 8 also inhibited the TGF-ß-induced nuclear translocation of SMAD with an EC(50) value of 0.8 µM. Kinetic analysis revealed that compound 8 inhibited ALK5 via mixed-type inhibition, suggesting that it may bind to ALK5 differently than other published adenosine triphosphate site inhibitors.


Assuntos
Ensaios de Triagem em Larga Escala , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Difosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Simulação por Computador , Transferência Ressonante de Energia de Fluorescência , Fluorimunoensaio , Humanos , Cinética , Conformação Molecular , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas Smad/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Fator de Crescimento Transformador beta/farmacologia
4.
Biochim Biophys Acta ; 1789(5): 422-31, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19414071

RESUMO

Histone deacetylase 5 (HDAC5) represses expression of nuclear genes that promote cardiac hypertrophy. Agonism of a variety of G protein coupled receptors (GPCRs) triggers phosphorylation-dependent nuclear export of HDAC5 via the CRM1 nuclear export receptor, resulting in derepression of pro-hypertrophic genes. A cell-based high-throughput screen of a commercial compound collection was employed to identify compounds with the ability to preserve the nuclear fraction of GFP-HDAC5 in primary cardiomyocytes exposed to GPCR agonists. A hit compound potently inhibited agonist-induced GFP-HDAC5 nuclear export in cultured neonatal rat ventricular myocytes (NRVMs). A small set of related compounds was designed and synthesized to evaluate structure-activity relationship (SAR). The results demonstrated that inhibition of HDAC5 nuclear export was a result of compounds irreversibly reacting with a key cysteine residue in CRM1 that is required for its function. CRM1 inhibition by the compounds also resulted in potent suppression of cardiomyocyte hypertrophy. These studies define a novel class of anti-hypertrophic compounds that function through irreversible inhibition of CRM1-dependent nuclear export.


Assuntos
Cardiomegalia/tratamento farmacológico , Histona Desacetilases/metabolismo , Carioferinas/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Amidas/farmacologia , Compostos de Anilina/farmacologia , Animais , Cardiomegalia/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Inibidores de Histona Desacetilases , Histona Desacetilases/química , Humanos , Carioferinas/metabolismo , Microscopia de Fluorescência , Miócitos Cardíacos/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade , Proteína Exportina 1
5.
Assay Drug Dev Technol ; 6(6): 811-8, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19231942

RESUMO

For ultra-high-throughput screening, 10-30 nl of compound dissolved in 75% dimethyl sulfoxide (DMSO)/25% water (vol/vol) is spotted into 1,536- and 3,456-well ChemLib plates (Aurora Biotechnologies, Carlsbad, CA) and stored appropriately for a short time before screening. Although this practice eliminates the compound plating bottleneck, plated volumes of DMSO slowly evaporate from assay wells if plates are not properly stored in the interim. Since many assays are sensitive to DMSO concentrations, even slight evaporation may cause intra-plate variation and thus decrease assay quality. Using a cytochrome P450 3A4 Vivid Blue assay (Invitrogen, Carlsbad), we investigated the rate, pattern, and quantity of evaporation over a 1-year time frame to identify best practices for long-term (i.e., 6 months or greater) storage of assay-ready compound plates. Our findings regarding evaporation at plate edges indicate that nanospots preplated in ChemLib 1,536- or 3,456-well plates are best stored at -80 degrees C, in a bag, with or without the outer evaporation wells filled or at -20 degrees C, in a bag, with evaporation wells filled.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Fluorometria/instrumentação , Fluorometria/métodos , Preservação Biológica/métodos , Bioensaio , Citocromo P-450 CYP3A/metabolismo , Dimetil Sulfóxido/química , Dimetil Sulfóxido/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/tendências , Corantes Fluorescentes/química , Umidade , Indicadores e Reagentes/química , Nanosferas/análise , Nanosferas/química , Refrigeração , Robótica , Solventes/química , Temperatura , Fatores de Tempo , Volatilização
6.
J Biomol Screen ; 11(7): 736-42, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16928980

RESUMO

Elongation Factor P (EF-P) is an essential component of bacterial protein synthesis, enhancing the rate of translation by facilitating the addition of amino acids to the growing peptide chain. Using purified Staphylococcus aureus EF-P and a reconstituted Escherichia coli ribosomal system, an assay monitoring the addition of radiolabeled N-formyl methionine to biotinylated puromycin was developed. Reaction products were captured with streptavidin-coated scintillation proximity assay (SPA) beads and quantified by scintillation counting. Data from the assay were used to create a kinetic model of the reaction scheme. In this model, EF-P binding to the ribosome essentially doubled the rate of the ribosomal peptidyl transferase reaction. As described here, EF-P bound to the ribosomes with an apparent K(a) of 0.75 microM, and the substrates N-fMet-tRNA and biotinylated puromycin had apparent K(m)s of 19 microM and 0.5 microM, respectively. The assay was shown to be sensitive to a number of antibiotics known to target ribosomal peptide bond synthesis, such as chloramphenicol and puromycin, but not inhibitors that target other stages of protein synthesis, such as fusidic acid or thiostrepton.


Assuntos
Antibacterianos/análise , Antibacterianos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Testes de Sensibilidade Microbiana , Fatores de Alongamento de Peptídeos/antagonistas & inibidores , Peptidil Transferases/antagonistas & inibidores , Proteínas Ribossômicas/antagonistas & inibidores , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Cinética , Reprodutibilidade dos Testes , Ribossomos/metabolismo , Staphylococcus aureus/metabolismo , Fatores de Tempo
7.
J Biomol Screen ; 8(3): 239-46, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12857377

RESUMO

High-throughput screening (HTS) has become an essential part of the drug discovery process. Due to the rising requirements for both data quality and quantity, along with increased screening cost and the demand to shorten the time for lead identification, increasing throughput and cost-effectiveness has become a necessity in the hit identification process. The authors present a multiplexed HTS for 2 nuclear receptors, the farnesoid X-activated receptor and the peroxisome proliferator-activated receptor delta in a viable cell-based reporter gene assay. The 2 nuclear receptors were individually transfected into human hepatoma cells, and the transient transfected cell lines were pooled for the multiplexed screen. Hits identified by the multiplexed screen are similar to those identified by the individual receptor screens. Furthermore, the multiplexed screen provides selectivity information if ligands selective for one and not the other receptor are one of the hit criteria. The data demonstrate that multiplexing nuclear receptors can be a simple, efficient, cost-effective, and reliable alternative to traditional HTS of individual targets without compromising data quality.


Assuntos
Núcleo Celular/metabolismo , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Genes Reporter , Linhagem Celular Tumoral , Sobrevivência Celular , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Humanos , Ligantes , Luciferases/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Transfecção
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